ES2968898T3 - In vitro method to detect kidney disease - Google Patents

Abstract

The present invention relates to the in vitro use of TRAF3 protein level determined in peripheral blood mononuclear cells (PBMC) isolated from blood samples obtained from the patient to detect kidney disease, to predict the response of patients suffering from kidney disease to a treatment. with vitamin analogues or derivatives of vitamin D, or to decide or recommend the treatment of patients suffering from kidney diseases with analogues or derivatives of vitamin D. (Automatic translation with Google Translate, without legal value)

Description

DESCRIPTION

In vitro method to detect kidney disease

field of invention

The present invention relates to the medical field. In particular, the present invention relates to the in vitro use of TRAF3 protein levels, determined in peripheral blood mononuclear cells (PBMC) isolated from blood samples obtained from healthy donors and patients, to detect kidney disease, to predict the response of patients suffering from kidney disease to treatment with vitamin D analogues or derivatives, or to decide or recommend whether patients suffering from kidney disease should be treated with vitamin D analogues or derivatives.

State of the art

Chronic kidney disease (CKD) is a type of kidney disease in which there is a gradual loss of kidney function over a period of months or years. In an early stage, there are usually no symptoms. However, at a later stage, swelling of the legs, feeling tired, vomiting, loss of appetite or confusion may develop. Complications may include heart disease, high blood pressure, bone disease, or anemia.

Causes of CKD include diabetes, hypertension, glomerulonephritis, and polycystic kidney disease. Risk factors include a family history of the condition. Diagnosis is usually made by blood tests to measure glomerular filtration rate and urine tests to measure albumin. Additional tests, such as an ultrasound or kidney biopsy, may be done to determine the underlying cause. Screening is recommended for people at risk. Initial treatments may include medications to control blood pressure, blood sugar levels, and lower cholesterol. NSAIDs should be avoided. Other recommended measures include staying active and certain dietary changes. Severe disease may require replacement therapies such as hemodialysis, peritoneal dialysis, or a kidney transplant. Treatments may also be needed for anemia and bone disease.

CKD affected 753 million people worldwide in 2016, including 417 million women and 336 million men. In 2015 it caused 1.2 million deaths, compared to 409,000 in 1990. The causes that contribute to the highest number of deaths are high blood pressure with 550,000, followed by diabetes with 418,000 and glomerulonephritis with 238,000.

The diagnosis of CKD is largely based on history, examination, and urine dipstick: glomerular filtration and/or albuminuria normalized to urinary creatinine levels or concentration. It is important to differentiate CKD from acute kidney injury (AKI) because AKI can be reversible. A diagnostic clue that helps differentiate CKD from AKI is a gradual increase in serum creatinine (over several months or years) rather than a sudden increase in serum creatinine (several days or weeks). In many patients with CKD, previous kidney disease or other underlying diseases are already known. A significant number of patients have CKD of unknown cause. Patients can progress from CKD to end-stage renal disease (ESRD).

In addition to controlling other risk factors, the goal of therapy is to slow or stop the progression of CKD. Control of blood pressure and treatment of the original disease are the general principles of treatment.

Nowadays, the definition of reliable biomarkers of kidney disease progression and its effective treatment is an unmet clinical need.

Reference [TRAF3 delays cyst formation induced by NF-KB signaling. Sun L, Hu C, Zhang X. IUBMB Life. 2017 March;69(3):170-178. doi: 10.1002/iub.1601. Epub of February 10, 2017], but the approach disclosed therein clearly differs from the present invention. Sun L.,et al.2017 only provide in vitro experiments in a transformed cell line in which the TRAF3 protein is overexpressed to determine its association with cell proliferation and apoptosis, both characteristics of polycystic kidney disease. Please note that kidney disease, particularly CKD, discussed in the present invention may be considered a different medical indication compared to polycystic kidney disease. It should be noted that CKD is characterized by inflammation and fibrosis, while apoptosis has not been reported in human CKD. In fact, renal polycytosis is a very specific inherited kidney disease that has an autosomal dominant genetic etiology. This occurs due to mutations in the PKD1 or PKD2 genes, which code for the proteins polycystin-1 and polycystin-2 (PC-1 and PC-2). On the other hand, note that Sun L.,et al.2017 do not provide reliable and reproducible conclusions. It is important to consider that in Sun L.,et al.2017, routine controls such as isolated transfection reagents or empty vectors have not been used, so the results provided in this publication regarding TRAF3 overexpression may not be conclusive. It is also important to note that, although TRAF3 protein levels are measured in CKD patients in the present invention, renal TRAF3 levels in injured kidneys are not determined in Sun L.,et al.2017. Last but not least, please note that minimally invasive samples are used in the present invention because TRAF3 levels are determined in blood cells obtained from CKD patients. Consequently, the method described in the present invention is a non-invasive procedure, which allows patients to be monitored by measuring TRAF3 levels over time. However, Sun L.,et al.

2017 is completely silent about the possibility of using blood samples.

Thus, the present invention focuses on providing a solution to a clear unmet medical need, which is the discovery of reliable biomarkers of the progression of kidney disease and the effective treatment thereof. The present invention solves this problem by using TRAF3 protein levels, determined in peripheral blood mononuclear cells (PBMC) isolated from blood samples obtained from the patient, to detect kidney disease, to predict the response of patients suffering from kidney disease to treatment with vitamin D analogues or derivatives, or to decide or recommend whether patients suffering from kidney disease should be treated with vitamin D analogues or derivatives.

Description of the invention

Brief description of the invention

The present invention clearly shows (see examples 1 and 2) that TRAF3 protein levels are significantly reduced in patients suffering from kidney disease in different scenarios: 1) in peripheral blood mononuclear cells (PMBC) from patients with ESRD versus healthy subjects , 2) in kidney tissue from animal models in which kidney damage was induced by the administration of TWEAK, folic acid (FA) or unilateral ureteral obstruction (UUO) (3 different and accepted models of experimental CKD) and 3) in vitro studies developed in tubular epithelial cells (representative renal cells of human [HK2] or murine [MCT] origin) and peripheral blood mononuclear cells (PMBC) in which renal damage was induced by TWEAK administration. Furthermore, the present invention also demonstrates that the use of vitamin D analogues or derivatives, for example, paricalcitol, results in an increase in TRAF3 protein levels which causes a positive therapeutic response in TWEAK-induced kidney damage. other types of models of acute kidney injury (AKI) or chronic kidney disease (CKD). In this case, a reduction in the expression of proinflammatory markers associated with kidney damage was observed, including a novel therapeutic modulation of the NF-kB2 signaling pathway.

Particularly, the first embodiment described herein relates to an in vitro method for determining the level of TRAF3 protein in a patient comprising: a) isolating peripheral blood mononuclear cells (PBMC) from blood samples obtained from the patient, and b ) determine the level of TRAF3 by using reagents capable of detecting TRAF3.

The second embodiment of the present invention refers to an in vitro method to detect kidney disease that comprises: a) determining at least the level of TRAF3 protein following the method of the first embodiment, and b) in which if the level determined in step ( a) is statistically lower than the level determined in healthy control subjects, this is an indication that the patient suffers from kidney disease.

The third embodiment of the present invention refers to an in vitro method to predict the response of patients suffering from kidney disease to treatment with analogues or derivatives of vitamin D that comprises: a) determining at least the level of TRAF3 protein following the first embodiment after administration of the vitamin D analogue or derivative, and b) in which if the level determined in step (a) is statistically higher than the expression level determined before administration of the vitamin D analogue or derivative, this It is an indication that the patient responds to treatment.

The fourth embodiment of the present invention relates to an in vitro method to decide or recommend whether patients suffering from kidney disease should be treated with analogues or derivatives of vitamin D comprising: a) determining at least the level of TRAF3 protein following the first embodiment, and b) wherein if the level determined in step (a) is statistically lower than the level determined in healthy control subjects, treatment with vitamin D analogues or derivatives is recommended.

The sixth embodiment of the present invention relates to the in vitro use of the TRAF3 protein level determined in peripheral blood mononuclear cells (PBMC) isolated from blood samples obtained from the patient to detect kidney disease, to predict the response of patients suffering from kidney disease to treatment with vitamin D analogues or derivatives, or to decide or recommend whether patients suffering from kidney disease should be treated with vitamin D analogues or derivatives.

The seventh embodiment that is not part of the present invention relates to a kit suitable for measuring the level of TRAF3 in a patient comprising: a) tools for isolating peripheral blood mononuclear cells (PBMC) from blood samples obtained from the patient; and b) reagents capable of detecting TRAF3.

The eighth embodiment that is not part of the present invention refers to the use of the kit defined above to detect kidney disease, to predict the response of patients suffering from kidney disease to treatment with analogues or derivatives of vitamin D, or to decide or recommend whether patients suffering from kidney disease should be treated with vitamin D analogues or derivatives.

In a preferred embodiment, the kidney disease manifests clinically as acute kidney injury (AKI) or chronic kidney disease (CKD), or the kidney disease has progressed to end-stage renal disease (ESRD). For the interpretation of the aforementioned terms LRA, ERC and ERT, please consult the following scientific publication:

[The Spanish Society of Nephrology (SENEFRO) commentary to the Spain GBD 2016 report: Keeping chronic kidney disease out of sight of health authorities will only magnify the problem. Ortiz A, Sanchez-Niño MD, Crespo-Barrio M, De-Sequera-Ortiz P, Fernández-Giráldez E, García-Maset R, Macía-Heras M, Pérez-Fontán M, Rodríguez-Portillo M, Salgueira-Lazo M, Sánchez-Álvarez E, Santamaría-Olmo R, Simal-Blanco F, Pino-Pino MD. Nephrology. JanFebruary 2019;39(1):29-34.doi: 10.1016/j.nefro.2018.09.002. Epub dated November 28, 2018].

For the purposes of the present invention, the following terms are defined:

• The term “comprising” means including, but not limited to, everything that follows the word “comprising.” Therefore, use of the term “comprising” indicates that the elements listed are required or mandatory, but that other elements are optional and may or may not be present.

• “Consisting of” is understood to include, and is limited to, what follows the expression “consisting of.” Therefore, the expression “consisting of” indicates that the elements listed are required or mandatory and that no other elements can be present.

• A “reference” value can be a threshold value or a limit value. Typically, a “threshold value” or “limit value” can be determined experimentally, empirically, or theoretically. A threshold value may also be arbitrarily selected based on existing experimental and/or clinical conditions, as one of ordinary skill in the art would recognize. The threshold value should be determined to obtain the optimal sensitivity and specificity according to the function of the test and the benefit/risk balance (clinical consequences of false positives and false negatives). Preferably, the person skilled in the art can compare the TRAF3 levels obtained according to the method of the invention with a defined threshold value. Additionally, retrospective measurement of TRAF3 levels (or scores) in properly stored historical samples of subjects can be used to establish these threshold values. The term "reference control level", according to the present invention, can refer to two different situations:

o“TRAF3 level determined in patients suffering from kidney disease”: In this sense, the patient would have been successfully treated with vitamin D analogues or derivatives if the TRAF3 level after the administration of the treatment is statistically higher compared to the level determined in control patients suffering from kidney disease.

o“TRAF3 level determined in healthy control subjects”: In this sense, patients would be identified as patients suffering from kidney disease, and consequently selected to receive treatment with a vitamin D analogue or derivative, if their level of TRAF3 is statistically lower compared to the level determined in healthy control subjects. •

• By “therapeutically effective dose or amount” of a composition comprising vitamin D analogs or derivatives is meant an amount that, when administered as described herein, produces a positive therapeutic response in a subject having kidney disease. . The exact amount required will vary from subject to subject, depending on the species, age and general condition of the subject, the severity of the condition being treated, the mode of administration and the like. One of ordinary skill in the art can determine an appropriate “effective” amount in any individual case using routine experimentation, based on the information provided herein.

Brief description of the figures

Figure 1. Paricalcitol restored TRAF3 in various kidney injury models. Evaluation of TRAF3 levels in experimental kidney damage, assessed by Western immunoblotting. A. Mice were treated with 750 ng/kg/day paricalcitol beginning 48 hours before administration of 0.5 μg TWEAK and sacrificed 24 hours later. B. Mice were treated with 25 ug/kg/day paricalcitol, starting 24 hours before 300 mg/kg folic acid (FA) or vehicle (0.3 mol/l sodium bicarbonate), and were studied after 24 hours. C. Mice were treated with 750 ng/kg/day paricalcitol, starting 48 hours before unilateral ureteral obstruction (UUO) and studied after 5 days. D. Paricalcitol restored TRAF3 levels in cytokine-stimulated cells. Human tubular cells (HK2) (D) or PBMC from healthy donors (E) were pretreated with paricalcitol (15 umol/L) 48 hours before stimulation with TWEAK (100 ng/mL) for 24 hours. TRAF3 protein levels by Western immunoblotting. Data expressed as mean ± SEM of 6-8 animals per group or 3 in vitro experiments. *p<0.05 versus control; #p<0.05 versus ERT cells or cells from injured kidney or treated with TWEAK.

Figure 2. Overexpression of TRAF3 mimics the actions of paricalcitol. TRAF3 overexpression was achieved in cultured cells using a TRAF3/CRISPR/Cas9 activating DNA plasmid. HK2 cells were stimulated with 100 ng/ml recombinant human soluble TWEAK. In some experiments, cells were preincubated for 48 hours with 15 pmol/L paricalcitol before stimulation with TWEAK. A. TRAF3 mRNA levels are increased in cells transfected with the TRAF3 CRISPR/Cas9 activation plasmid as assessed by real-time PCR. B. Activation of the NF-<k>B2 pathway was assessed by Western blotting of the p52 subunit of NFkB2 and the NFkB2-regulated cytokine CCL21A. C and D. Gene expression in MCT of the proinflammatory factors CCL2, CCL5 and IL-6 (C) or CCL21A and CCL19 (D) was evaluated by real-time PCR. Data expressed as mean ± SEM of 3-5 independent experiments. *p<0.05 versus control; #p<0.05 versus cells treated with TWEAK.

Figure 3. Paricalcitol restored TRAF3 levels in ESRD patients and in cultured peripheral blood mononuclear cells (PBMCs). A. TRAF3 protein levels in PBMC from ESRD patients treated or not with paricalcitol (15 umol/L) were determined by Western immunoblotting. Number of patients 5-8 per group. *p<0.05 versus control; #p<0.05 versus ERT cells. B. TRAF3 mRNA levels in PBMC from ESRD patients treated or not with paricalcitol were determined by real-time RT-PCR. Number of patients 5-8 per group. *p<0.05 versus control. Number of patients 5 per group. *p<0.05 versus control.

Figure 4. Modulation of TRAF3 ubiquitination and CIAP1 complex formation was involved in the restoration of TRAF3 levels with paricalcitol. A. In PBMCs from ESRD on hemodialysis, treated or not with the vitamin D receptor activator (VDRA) paricalcitol, TRAF3 was immunoprecipitated (IP) using an anti-TRAF3 antibody followed by SDS-PAGE and type immunoblotting was performed. Western (IB) against anti-ubiquitin-Lys-48, TRAF3 and CIAPi antibodies to analyze the possible link between them. Representative experiment. Antibody against TRAF3 was used as a loading control. Number of patients 5-8 per group. B. TWEAK-induced TRAF3 ubiquitination was prevented with paricalcitol in HK2 cells stimulated with TWEAK (100 ng/ml) and treated or not with paricalcitol (15 umol/l). TRAF3 was immunoprecipitated (IP) using anti-TRAF3 antibody followed by SDS-PAGE and western immunoblotting (IB) was performed against anti-ubiquitin-Lys-48, TRAF3, and CIAP1 antibodies to analyze the possible link between them. Representative experiment. Antibody against TRAF3 was used as a loading control. The figures show a representative PI experiment of the 3 carried out.

Figure 5. Paricalcitol (VDRA) inhibits the non-canonical NF-<k>B signaling pathway (NF-<k>B2), but not the canonical NF-<k>B signaling pathway (NF-<k>B). >B1) in PBMC from patients with end-stage renal disease (ESRD) on hemodialysis. Activation of canonical and noncanonical NF-<k>B pathways in PBMC was assessed by the DNA binding activities of p65 (A) or p52 and RelB (B), respectively. Number of patients: 5-8 per group. *p<0.05 versus healthy control; #p<0.05 versus patients with ESRD without paricalcitol. (C) Paricalcitol decreased proinflammatory genes in PBMC from end-stage renal disease (ESRD) patients on hemodialysis. PBMCs were isolated from control individuals or ESRD patients with and without paricalcitol and analyzed for proinflammatory gene expression. Gene expression was evaluated by real-time PCR. Number of patients: 5-8 per group.

Figure 6. Paricalcitol decreased non-canonical NF-<k>B2 activation but not canonical NF-<k>B activation induced by TWEAK in renal tubular epithelial cells and in PBMC from healthy donors. A-D. Human tubular cells were pretreated with 12 pM paricalcitol for 48 hours before stimulation with 100 ng/ml TWEAK for 15 minutes (NF-<k>B1 pathway) or 6 hours (NF-<k>B2 pathway). ). Canonical NF-<k>B1 activation was assessed by phosphorylation of kBa or p65 in total protein extracts and p65 levels in cytosolic or nuclear fractions (A and B). Activation of the non-canonical NF-<k>B2 pathway was assessed by assessing the levels of NIK and phosphorylated IKK-α in total protein extracts (A and C) and the subcellular localization of the p100/p52 subunits and Rel B (A and D). GAPDH and Histone H1 were used as total and nuclear protein loading controls, respectively. A. Representative Western blot. B, C and D. Quantification of protein levels expressed as mean ± SEM of 4 experiments. *p<0.05 versus control. # p<0.05 vs. TWEAK. E. Human PBMC cells were pretreated with 12 pM paricalcitol for 24 hours, before stimulation with 100 ng/ml TWEAK (E). Canonical NF-<k>B1 activation was assessed as phosphorylated p65 in cytosolic fractions and non-canonical NF-<k>B2 activation was assessed by p52 levels. *p<0.05 versus control; #p<0.05 vs stimulated with TWEAK.

Figure 7. Paricalcitol decreased proinflammatory genes, cytokine-stimulated PBMCs from control individuals or tubular cells in culture. A. PBMCs were isolated from control individuals with and without paricalcitol and analyzed for proinflammatory gene expression. B. PBMC were isolated from control individuals and stimulated in culture with cytokines. C. Murine cultured tubular epithelial cells. PBMC or cultured murine tubular epithelial cells were stimulated with 100 ng/ml TWEAK for 6 hours. Cells were preincubated for 48 hours with 15 pmol/L paricalcitol or 60 ng/mL SN52. Gene expression was evaluated by real-time PCR. Number. Data expressed as mean ± SEM of 3-6 experiments. *p<0.05 versus control; #p<0.05 versus cells treated with TWEAK.

Figure 8. Paricalcitol or NF-kB2 blockade ameliorated TWEAK-induced renal inflammation. C57BL/6 mice were treated with 750 ng/kg/day paricalcitol or 0.7 mg/day of the NF-<k>B2 inhibitor SN52 (two doses; day -1; day 0) starting 48 hours before 0. 5 |jg of TWEAK, and were sacrificed 24 hours after TWEAK administration. A. Immunohistochemistry using anti-F4/80 and anti-CD3 antibodies identified monocytes/macrophages and T lymphocytes, respectively. Representative animal of each group. 200X magnification. B. Quantification of staining C. RNA was obtained from total kidney extracts and the expression levels of pro-inflammatory genes (CCL2, CCL5 and IL6) were determined by real-time PCR. D. Renal CCL2 and CCL5 protein levels were assessed by ELISA. E. Gene expression levels of the NF-kB2-regulated cytokines CCL21A and CCL19 were determined by real-time PCR. Data expressed as mean ± SEM of 8-10 animals per group. *p<0.05 versus control. # p<0.05 vs. TWEAK.

Figure 9. Paricalcitol alone inhibits non-canonical NF-<k>B activation induced by TWEAKin vivo. Mice were treated with 750 ng/kg/day paricalcitol starting 48 hours before 0.5 μg of TWEAK and sacrificed. 24 hours after TWEAK administration. A and B. In TWEAK-injected mice, elevated cytosolic phosphorylated kBa (A) and nuclear p65 levels (B) were found but were not modulated by paricalcitol treatment, suggesting that paricalcitol did not modulate activation. of NF-<k>B1. GAPDH and Ponceau red were used as loading controls. Data expressed as mean ± SEM of 8-10 mice per group. *p<0.05 versus control. C. Nuclear localization of p52 and Rel B in renal tubules of TWEAK-injected mice assessed by immunohistochemistry. D. Western blot evaluation of NF-kB2 activation (p52 levels) in whole kidney protein extracts. E. In renal nuclear protein extracts, the DNA binding activity of p52 and RelB of NF-kB2 was measured by ELISA. All data expressed as mean ± SEM of 8-10 animals per group. *p<0.05 versus control. # p<0.05 vs. TWEAK.

Figure 10. Paricalcitol inhibits TWEAK-induced upregulation of specific NF-<k>B2 targets in the kidney. Mice were treated with 750 ng/kg/day paricalcitol or 0.7 mg/day of the NF-κB2 inhibitor SN52 (two doses; day -1; day 0) starting 48 hours before 0.5 μg of TWEAK, and were sacrificed 24 hours after TWEAK administration. A. RNA was obtained from total kidney extracts and gene expression of the NF-kB2-regulated cytokines CCL21A and CCL19 was determined by real-time PCR. B. CCL21A protein levels were assessed by Western blotting on total kidney proteins. GAPDH was used as a loading control. C. Expression of CCL21A in the kidney. Representative mouse of each group. D. Quantification of CCL21A-stained area versus total area. Data expressed as mean ± SEM of 8-10 mice per group. *p<0.05 versus control. # p<0.05 vs. TWEAK.

Figure 11. The SN52 peptide blocks the activation of NF-kB2 and the nuclear translocation of p52 in a TWEAK-induced kidney injury model. Mice were pretreated with 0.7 mg/day of NF-<k>B2 inhibitor SN52 (two doses; day -1; day 0) starting 48 hours before administration of 0.5 μg of TWEAK, and sacrificed 24 hours after TWEAK administration. A. Immunohistochemistry of p52 in paraffin-embedded kidney sections. Representative animal of each group (200x magnification). B and C. Western blot for p52 (B) and kBa (C) phosphorylation. Data expressed as mean ± SEM of 8-10 animals per group. *p<0.05 versus control. # p<0.05 vs. TWEAK.

Figure 12. Paricalcitol inhibits the activation of NF-<k>B2, but not NF-<k>B1 in experimental unilateral ureteral obstruction (UUO) in mice. Mice were treated with 750 ng/kg/day paricalcitol, starting 24 hours before UUO, and were studied 5 days after UUO. A. Immunohistochemistry in paraffin-embedded kidney sections. Representative animal of each group (200x magnification). B. Western blot, NF-kB2 activation was assessed as p52 levels in total protein extracts. C. In isolated renal nuclear proteins, the DNA binding activities of p52 and RelB were measured by ELISA. D and E. Western blot assessment of nuclear levels of p65 phosphorylation (D) and cytosolic kBa (E) was used to assess activation of the NF-<k>B1 pathway. In each mouse, the obstructed kidneys (Ob) were compared with the corresponding contralateral non-obstructed kidney (Nob). Data expressed as mean ± SEM of 6-8 animals per group. *p<0.05 versus contralateral #p<0.05 versus obstructed kidneys.

Figure 13. Paricalcitol decreases renal inflammation in experimental unilateral ureteral obstruction (UUO) in mice. Mice were treated with 750 ng/kg/day paricalcitol, starting 24 hours before UUO, and were studied 5 days after UUO. In paraffin-embedded kidney sections, immunohistochemistry using anti-F4/80 and anti-CD3 antibodies identified monocytes/macrophages and T lymphocytes, respectively. A. Representative animal of each group. 200X magnification. B, C. In the RNA obtained from total kidney extracts, the expression of proinflammatory genes (CCL2, CCL5, IL-6, TNF-a and IL-17A) and the expression of specific genes regulated by NF<k>B2 (CCL19 and CCL21) were determined by real-time PCR. Data expressed as mean ± e Em of 6-8 animals per group. *p<0.05 compared to the contralateral non-obstructed kidney (NOb); #p<0.05 versus obstructed kidneys (Ob).

Figure 14. Paricalcitol inhibits the activation of NF-<k>B2, but not that of NF-<k>B1 in folic acid-induced kidney injury. Mice were treated with 25 pg/kg/day paricalcitol starting 24 hours before administration of 300 mg/kg folic acid (FA) or vehicle (0.3 mol/L sodium bicarbonate), and were studied 24 hours after AF injection. A. Immunohistochemistry revealed nuclear localization of p52 and RelB that was decreased by paricalcitol. Representative animal of each group (200x magnification). B and C. Western blot of p52, as evidence of NF-<k>B2 activation (B) and kBa phosphorylation as evidence of NF-<k>B1 activation (C). D. CCL2 protein levels assessed by ELISA. E. In isolated renal nuclear proteins, the DNA binding activity of RelB was assessed by ELISA. F. CCL21 protein levels assessed in total kidney protein extracts by Western blotting. G. RNA was obtained from total kidney extracts and expression levels of pro-inflammatory genes were determined by real-time PCR. H. Data on serum BUN levels are shown. Data expressed as mean ± SEM of 6-8 animals per group. *p<0.05 versus control; #p<0.05 versus folic acid kidneys.

Figure 15. Paricalcitol decreases renal inflammation induced by folic acid in mice. Mice were treated with 25 pg/kg/day paricalcitol, starting 24 hours before 300 mg/kg folic acid or vehicle (0.3 mol/L sodium bicarbonate), and were studied 24 hours after administration of folic acid. Immunohistochemistry on paraffin-embedded kidney sections using anti-F4/80 and anti-CD3 antibodies identifying monocytes/macrophages and T lymphocytes, respectively. The figure shows a representative animal from each group. 200X magnification.

Figure 16. Paricalcitol inhibits TWEAK-induced upregulation of specific NF-<k>B2 targets in cells with the MARSS gene silenced (another possible vitamin D receptor). MARSS gene silencing was achieved in cultured cells using a predesigned and validated siRNA against MARSS. Cells were stimulated with 100 ng/ml recombinant human soluble TWEAK. In some experiments, cells were preincubated for 48 hours with 15 pmol/L paricalcitol before stimulation with TWEAK. A. Activation of the NF-<k>B2 pathway was assessed by Western blotting of NF<k>B2 p52. Data expressed as mean ± SEM of 3-5 independent experiments. *p<0.05 versus control; #p<0.05 versus cells treated with TWEAK.

Detailed description of the invention

Example 1. Material and methods.

Example 1.1 Experimental models.

All animal procedures were performed in accordance with the animal research guidelines in the European Community and with prior approval from the IIS-Fundación Jiménez Díaz Health Research Ethics Committee. For in vivo studies, mice received a daily intraperitoneal injection of VDRA Paricalcitol beginning 48 hours before induction of kidney damage.

At sacrifice, animals were anesthetized with 5 mg/kg xylazine (Rompun, Bayer AG) and 35 mg/kg ketamine (Ketolar, Pfizer) and the kidneys were perfused in situ with cold saline before removal. Kidney slices were then fixed in formalin buffer for immunohistochemistry studies or immediately frozen in liquid nitrogen for gene and protein studies.

TWEAK administration: C57BL/6 female mice (9-12 weeks, weight 20 g, 7-8 animals per group) received a single intraperitoneal injection of 0.5 pg of TWEAK dissolved in saline and were sacrificed 24 hours later, as previously described (Sanzet al.2008). Controls were injected with the saline vehicle (n = 8-10 mice per group). The endotoxin level of TWEAK was <0.1 ng/mg, confirmed by MALDI-TOF. Some animals received 750 ng/kg/day paricalcitol or 0.7 mg/mouse/day NF-κB2 inhibitor SN52, starting 48 hours before TWEAK administration. The animals were sacrificed 24 hours later. The dose of paricalcitol was chosen based on previous experience with mild inflammatory kidney conditions.

Unilateral ureteral obstruction (UUO) was established in male C57BL/6 mice under isoflurane-induced anesthesia. The left ureter was ligated with silk (5/0) in two places and cut between ligations to prevent urinary tract infection (obstructed kidney). (Uceroet al.2014; Rodrigues-Díezet al.2013). Two groups were studied; untreated or treated with paricalcitol (750 ng/kg/day, i.p., Abbot), n=6-8 mice per group, starting 48 hours before surgery. The studies compared both kidneys (contralateral vs. obstructed) in each mouse. Folic acid nephropathy is a classic model of renal tubulointerstitial injury and inflammation that has been reported in humans. Mice received a single intraperitoneal injection of 300 mg/kg folic acid (Sigma) in 0.3 mol/L sodium bicarbonate or vehicle and were sacrificed 48 hours later. Two groups were studied; untreated or treated with paricalcitol (25 pg/kg/day, i.p., Abbot), n=7-8 per group starting 48 hours before FA injection. The dose of paricalcitol was chosen based on preliminary dose-finding experiments.

Example 1.2. Cultured cells.

Human kidney proximal tubule epithelial cells (HK2 cell line, ATCC CRL-2190) were cultured in RPMI 1640 with 10% fetal bovine serum (FBS), 1% glutamine, 100 U/ml penicillin, 100 μg/ml of streptomycin, 5 |jg/ml of insulin-transferrin-selenite and 36 ng/ml of hydrocortisone in 5% CO2 at 37 °C. When the cells reached 60 to 70% confluency, they were serum starved for 24 hours before the experiment.

Murine proximal tubulo-epithelial cells (MCT cell line) were originally obtained from Dr. Eric Neilson (Vanderbilt University) and used for gene expression studies. These cells were cultured in RPMI 1640 with 10% FBS, 1% glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin in 5% CO2 at 37°C. When cells reached 60 to 70% confluency, they were maintained in RPMI with 1% FBS for 24 hours.

Cells were stimulated with 100 ng/ml recombinant human soluble TWEAK (Millipore) or 200 μg/ml p-cresyl sulfate (Raymond Vanholder and Griet Glorieux, Gent University, Belgium). The concentrations of TWEAK and p-cresyl sulfate were based on previously published dose-response experiments (Povedaet al.2014; Sanzet al.2008). In some experiments, cells were preincubated for 48 hours with 12 μmol/L paricalcitol (Abbott) before stimulation. DMSO, used as a solvent in some cases, had no effect on cell viability or gene expression levels.

Example 1.3. PBMC isolation.

Intracellular protein and mRNA levels were measured in PBMC from ESRD patients on hemodialysis. The inclusion criteria were the following: age >45 years; male sex; informed consent; and absence of active inflammatory, infectious or malignant diseases at baseline or during the study. This study was approved by the Ethics Committee of the IIS-Fundación Jiménez Díaz. Patients were enrolled after providing written informed consent. PBMCs were separated from whole blood samples (30 ml in EDTA tubes) by a density gradient centrifugation method using Ficoll density gradient centrifugation. In total, 50x106 PBMCs were obtained from 30 ml of whole blood and 6x106 PBMCs were used to isolate protein, RNA, and nuclear/cytosolic protein levels using NE-PER reagent (Pierce) following the manufacturer's instructions.

Example 1.4. Renal histology and immunohistochemistry.

Paraffin-embedded kidney sections (3 μm) were stained using conventional methods. Antigen retrieval was performed using the PTlink link system (Dako Diagnostics) with sodium citrate buffer (10 mmol/l) adjusted to pH 6-9 depending on the immunohistochemical marker, followed by immunohistochemical staining on a Dako Autostainer device. Steps: 1) blockade of endogenous peroxidase; 2) incubation of primary antibodies; anti-CD3 (1:300; Dako) or anti-F4/80 (1:5000; Serotec) antibodies; NF-<k>B p-p65 (1:200; Cell Signaling); NF-<k>B2 p52 (1:50; Cell Signaling); RelB (1:50; Santa Cruz Biotechnology); CCL21 (1:90; Santa Cruz Biotechnology); 3) washing; 4) Treatment in DUOFLEX Doublestain EnVision™, using 3,3'-diaminobenzidine as chromogen. For F4/80 staining, a rabbit anti-rat IgG linker was used before EnVision. The sections were counterstained with Carazzi hematoxylin.

Reactive label intensity was obtained using Image-Pro Plus software. For each sample (processed in duplicate blindly), the average value was obtained from the 4-field analysis (20X objective) as density/mm2 or percentage of stained area versus total area analyzed. Data are expressed as fold increase relative to control mice, as mean ± SEM of 8-10 animals per group. Negative controls include nonspecific immunoglobulin and no primary antibody.

Example 1.5. Protein studies.

Proteins were obtained from treated cells or mouse kidneys using lysis buffer (50 mmol/l Tris-HCl, 150 mol/l NaCl, 2 mmol/l EDTA, 2 mmol/l EGTA, 0.2 mmol/l Triton %, IGEPAL 0.3%, proteinase inhibitor cocktail 10 μl/ml, PMSF 0.2 mmol/l and orthovanadate 0.2 mmol/l). The BcA method was used to determine the protein content.

For Western blotting, protein extracts from cells (25 μg/lane) and kidney (100-150 μg/lane) were separated on 6%-12% SDS-polyacrylamide gels under reducing conditions. Samples were then transferred to nitrocellulose membranes (BioRad), blocked with TBS/5% nonfat milk/0.05% Tween-20, and incubated overnight at 4°C with the following antibodies (dilution). : anti-p100/p52 of NF-<k>B2 (1:500), p-p65 of NF-<k>B (1:500) and p65 of NF-<k>B (1:500, Cell Signaling ); CCL-21A (sc-25445, 1:500); RelB (sc-226, 1:500); p-IKKa (sc-101706, 1:500); p-kBa (sc-8404, 1:500), kBa (sc-371, 1:500); TRAF3 (sc-6933, 1:250); CIAP-1 (sc-271419, 1:500); NIK (sc-7211, 1:500); VDR (sc-1008, 1:250, Santa Cruz Biotechnology). Subsequently, the membranes were incubated with peroxidase-conjugated IgG secondary antibody and revealed using an ECL chemiluminescence kit (Amersham). Loading controls included anti-GAPDH (1:10000; Chemicon), anti-α tubulin (1:5000; Sigma-Aldrich), or anti-histone H1 (sc-8030, 1:250, Santa Cruz Biotechnology) antibodies for proteins. nuclear or total protein levels in phosphorylation studies. Autoradiographs were scanned using the Gel Doc™ EZ imager and analyzed with Image Lab 3.0 software (BioRad).

ELISA was used to evaluate the levels of the chemokines CCL2 and CCL5 (eBioscience). In kidney samples, total protein content was determined by the BCA method and equal amounts of protein were analyzed. Data are expressed as an n-fold increase over the mean control levels.

Example 1.6. Gene expression studies.

Total RNA was isolated from mouse cells and kidney samples with Trizol (Invitrogen). cDNA was synthesized using the High Capacity cDNA Archive Kit (Applied Biosystems) using 2 μg of total RNA primed with random hexamer primers. Real-time multiplex C was performed using Applied Biosystems expression assays for murine and human samples: C2 Mm00441242_m1; CCL5 Mm_01302428_m1; IL-6 Mm_00446190_m1; CCL19 Mm_00839967_g1, CCL21A Mm_036466971_gH; TRAF3 Hs_00936781_m1; CCL19 -Hs_00171149_m1; CCL21A Hs_00989654_m1. Data were normalized to 18S; 4210893E (VIC) and GAPDH Mm99999915_g1. The mRNA copy numbers were calculated for each sample by the instrument software using the Ct value. Results are expressed in copy numbers, calculated relative to unstimulated cells or control mice, after normalization against 18S.

Example 1.7. ELISA-based NF-<k>B2 and RelB assay.

Nuclear and cytoplasmic fractions were separated from kidney tissues using NE-PER reagent (Pierce) following the manufacturer's instructions. In renal nuclear extracts, the DNA binding activity of NF-<k>B2 and RelB was measured by their binding to an oligonucleotide containing the NF-<k>B consensus site using an NF-<k>B family kit. >B TransAM (Active Motif) with an antibody that only recognizes p52 and RelB of active NF-<k>B2.

Example 1.8. Expression of magnetofection activation genes.

Gene overexpression was performed in cultured cells using the TRAF3CRISPR/Cas9 activation DNA plasmid (Santa Cruz Biotechnology). Subconfluent cells were transfected with magnetofection™ mixture (OZ biosciences) [PolyMag CRISPR magnetofection reagent (1 μl) plasmid DNA (1 μg)] for 30 minutes on the magnetic plate according to the manufacturer's instructions. Cells were then incubated with 10% heat-inactivated FBS for 24 h and incubated in serum-free medium for 24 h before experiments. Cells were stimulated with recombinant human soluble TWEAK (Millipore). In some experiments, cells were preincubated for 48 hours with 12 μmol/L paricalcitol (Abbott) before stimulation. Activation of the NF-<k>B2 pathway was assessed by gene expression and Western blot analysis using 1:200 NF-kB2 anti-p100/p52 and 1:200 CCL21A antibodies. Anti-TRAF3 antibody (1:250, Santa Cruz Biotechnology) was used to evaluate the specificity and efficiency of overexpression, and anti-GAPDH antibody (1:10000) was used as a loading control.

Example 1.9. Gene silencing.

Gene silencing in cultured cells was performed using a predesigned siRNA corresponding to VDR (Stealth Select RNAiTM, Ambion). Subconfluent cells were transfected for 24 h with 25 nmol/L siRNA using 50 nmol/L Lipofectamine RNAiMAX (Invitrogen, according to the manufacturer's instructions). Cells were then incubated with 10% heat-inactivated FBS for 24 h, followed by 24 h in serum-free medium.

Example 1.10. Coimmunoprecipitation assays.

Cells grown on tissue culture plates were lysed in 300-500 μl of Triton-NP-40 lysis buffer [50 mmol/l Tris-HCl, pH 8, 150 mmol/l NaCl, 1 mmol/l phenylmethylsulfonyl fluoride. l, 1% NP-40/IGEPAL, and a phosphatase inhibitor cocktail (Set II, Calbiochem)], were scraped from the plate and incubated for 1 h at 4 °C with shaking. Cell lysates were precleared by incubating with 10 μl of agarose and protein A bead suspensions (0.5 ml agarose/2 ml phosphate-buffered saline) for 30 min at 4°C and then centrifuged three times for 5 minutes at 2500 rpm to wash the supernatants. Previously cleared lysates were incubated with 2.5-5 μg of mouse monoclonal anti-TRAF3 antibody (sc-6933, Santa Cruz Biotechnology) overnight at 4 °C. Immune complexes were captured by adding protein A/G PLUS-agarose bead suspensions (20 μl) for 1 hour at 4°C. Agarose beads were collected by centrifugation, washed three times with lysis buffer, resuspended in 2x Laemmli sample buffer, boiled for 5 min, and subjected to SDS-PAGE. Western immunoblotting was then performed as described above, using specific anti-ubiquitin-Lys-48 antibodies 1:250 (#05-1307, Millipore) or anti-TRAF31:250 antibodies (sc-6933, Santa Cruz Biotechnology). Example 1.11. Statistic analysis.

All results are expressed as mean ± SEM. Differences between intervention groups and controls were evaluated using the Mann-Whitney test. P<0.05 was considered significant. Statistical analysis was performed using SPSS statistical software (version 11.0).

Example 2. Results.

• TRAF3 as a reliable biomarker of kidney damage.

Example 2.1. TRAF3 is downregulated in injured kidneys in different experimental models of kidney injury.

First, changes in TRAF3 protein levels were evaluated in different experimental models of kidney damage. In different experimental models of kidney injury in mice, including folic acid-induced kidney injury, TWEAK-mediated renal inflammation, and unilateral ureteral obstruction, TRAF3 levels were down-regulated in injured kidneys, as determined by immunoblotting. Western type in total kidney proteins (Figures 1A; 1B and 1C). Next, the direct effect of kidney damage mediators on TRAF3 modulation was evaluated. Studies were performed in human tubular epithelial cells cultured as representative renal cells expressing components of the non-canonical NF-<k>B2 pathway that promote kidney injury and in PBMC from healthy donors. Cells were stimulated with a tumor necrosis factor-like weak inducer of apoptosis (TWEAK), one of the few cytokines that can activate both NF-κB pathways. TWEAK stimulation downregulated TRAF3 levels in both kidney cells and PBMCs (Figures 1D and 1E).

Example 2.2. TRAF3 modulates the non-canonical NF-kB2 pathway and proinflammatory cytokines.

To investigate the functional consequences of TRAF3 modulation, cells were transfected with a TRAF3 CRISPR/Cas9 activation plasmid (Figure 2A). Overexpression of TRAF3 in HK2 cells blocked TWEAK-induced p52 activation (Figure 2B), as well as increased expression of proinflammatory genes such as CCL2, CCL5, and IL6 (Figure 2C), as well as genes regulated by NF-kB2 (Figure 2C). Figure 2D).

Example 2.3. TRAF3 is downregulated in circulating cells from patients with chronic kidney disease.

Next, TRAF3 levels in ESKD patients were investigated using PBMC, the main cell type involved in the pathogenesis of inflammatory diseases. In PBMCs from R<t>patients, TRAF3 protein was downregulated compared to healthy controls (Figure 3A), while TRAF3 mRNA levels were upregulated, suggesting that the Modulation of TRAF3 protein levels occurs through post-translational modifications (Figure 3B).

In summary, the preclinical data of the present invention show that, in response to kidney damage, TRAF3 levels are downregulated in cytokine-stimulated renal cells and PBMCs, as well as in injured murine kidneys. Furthermore, in circulating cells from ESRD patients, TRAF3 levels were also downregulated. All these data show that TRAF3 is a reliable biomarker of kidney damage.

Example 2.4. Downregulation of TRAF3 in circulating cells of patients with chronic kidney disease is related to progression to ESKD.

On the other hand, TRAF3 levels were investigated in two groups of patients with CKD, a group of patients who do not progress to ESRD and another group who progress. PBMC from CKD patients belonging to those groups were used. TRAF3 protein was clearly downregulated in samples from progressing patients compared to non-progressing patients.

In summary, the preclinical data of the present invention show that TRAF3 levels are downregulated in circulating cells of patients who progress to ESRD, compared to CKD patients who do not progress to ESRD. All these data show that TRAF3 is a reliable biomarker of kidney damage progression. •

• TRAF3 levels are modulated by therapeutic treatments with vitamin D analogues or derivatives.

Example 2.5. Evaluation of TRAF3 modulation by VDRA in patients with ESRD.

In PBMCs from ESRD patients treated with paricalcitol on hemodialysis, TRAF3 protein levels were restored to values in the range of healthy controls, while no changes were found in TRAF3 mRNA levels, suggesting that paricalcitol prevents TRAF3 degradation (Figures 3A, 3B).

Example 2.6. Evaluation of TRAF3 modulation by VDRA in cytokine-stimulated cells and injured kidneys.

In cultured tubular epithelial cells (HK2 cell line), preincubation with paricalcitol restored TWEAK-induced TRAF3 protein levels to values similar to those in untreated control cells (Figure 1D). Accordingly, in PBM<c>from healthy donors, paricalcitol inhibited TWEAK-induced TRAF3 degradation (Figure 1E).

In different experimental models of kidney injury in mice (folic acid-induced kidney injury, TWEAK-mediated kidney inflammation, and unilateral ureteral obstruction), daily treatment with paricalcitol, started one day before kidney damage, restored renal TRAF3 protein levels to the values of the control mice. (Figures 1A; 1B and 1C).

Next, the upstream mechanisms involved in TRAF3 degradation were investigated. In PBMCs from ESRD patients, TRAF3 immunoprecipitation studies showed increased TRAF3 ubiquitination on K48-linked chains, leading to the formation of CIAP1-TRAF3 complexes (Figure 4A). In patients treated with paricalcitol, TRAF3 ubiquitination and CIAP1-TRAF3 interaction were significantly decreased to values in the range of healthy controls (Figure 4A). Furthermore, CIAP-1 levels were higher in whole protein extracts of PBMC from ESRD patients than in healthy controls, while levels in ESRD patients treated with paricalcitol were close to control values (Figure 4A ). Similar results were observed in cultured cells stimulated by cytokines. In HK2 cells, TWEAK stimulation induced TRAF3 ubiquitination and CIAP1-TRAF3 complex formation and this was prevented by paricalcitol (Figure 4B).

Example 2.7. The modulation of TRAF3 by paricalcitol was related to the activation and inflammation of the non-canonical NF-kB2 pathway.

• VDRA paricalcitol inhibits activation of the non-canonical NF-kB2 pathway without modulating the canonical NF-kB1 pathway in PBMC from CKD patients.

In PBMCs from ESRD patients on hemodialysis, both canonical and non-canonical NF-<k>B pathways were activated, as demonstrated by increased DNA-binding activities of p65/NF-KB1 and RelB or p52/ NF-KB2 (Figures 5A and 5B). Interestingly, in PBMCs from ESRD patients treated with paricalcitol, activation of p65/NF-KB1 was similar to that in ESRD patients without paricalcitol, while activation of the NF-<k>B2 pathway was inhibited ( Figures 5A and 5B). These data indicate that paricalcitol alone inhibits noncanonical NF-κB2 activation.

• Paricalcitol decreases cytokine-induced proinflammatory cytokines in patients with ESRD.

In PBMCs from ESRD patients on hemodialysis, the expression levels of proinflammatory genes increased, but were significantly lower in ESRD patients treated with paricalcitol (Figure 5C). This observation supports the anti-inflammatory properties of paricalcitol in human CKD.

Example 2.8. Evaluation of the modulation of TRAF3 by VDRA in cells and its possible relationship with the regulation of NF-κB/inflammatory pathways.

• Paricalcitol alone regulates activation of the NF-KB2 pathway in cultured cells stimulated by cytokines In cultured tubular epithelial cells (HK2 cell line), TWEAK increased p65 phosphorylation and kBa phosphorylation and downregulated kBa levels. cytosolic (Figures 6A and 6B). Preincubation with paricalcitol did not modulate TWEAK-induced NF-<k>BI activation (Figures 6A and 6B), while paricalcitol inhibited TWEAK-induced IKK-α phosphorylation, increased nuclear levels of NIK, and p52. and RelB (Figures 6A, 6C and 6D). Thus, paricalcitol inhibited TWEAK-induced activation of the noncanonical NF-κB pathway, but did not modulate canonical NF-<k>B activation.

In PBMCs from healthy donors, paricalcitol inhibited TWEAK-induced NF-κB2 activation without modulating the canonical NF-κBI pathway (Figure 6E).

Paricalcitol's direct regulation of proinflammatory genes was confirmed in cultured cells. In PBMCs obtained from healthy donors, paricalcitol inhibited TWEAK-induced upregulation of several proinflammatory genes, such as CCL2, CCL5, and IL6, as well as the NF-KB2-controlled genes CCL21A and CCL19 (Figure 7A). Similar results were obtained in cultured tubular cells (Figure 7B and 7C). Furthermore, in tubular cells, inhibition of NF-KB2 with SN52 peptide prevented TWEAK-induced upregulation of proinflammatory genes (Figures 7B and 7c), mimicking the effects of paricalcitol.

Example 2.9. Evaluation of TRAF3 modulation by VDRA in preclinical experimental models of kidney damage and its possible relationship with the regulation of NF-KB/inflammatory pathways.

• Paricalcitol decreases TWEAK-induced renal inflammation in mice by modulating the activation of the non-canonical NF-kB2 pathway.

TWEAK administration elicits an acute inflammatory response in murine kidneys and activates both canonical and non-canonical NF-<k>B pathways. Therefore, a TWEAK-induced renal inflammation model was used to evaluate the effect of paricalcitol on NF-κB activation in the kidney. In TWEAK-injected mice, paricalcitol significantly decreased TWEAK-induced upregulation of infiltrating monocytes/macrophages (F4/80+ cells), T lymphocytes (CD3+ cells) (Figures 8A and 8B), and proinflammatory factors, such as CCL2, CCL5 and IL-6 (Figures 8C and 8D). In kidneys from TWEAK-injected mice, kBa phosphorylation and nuclear NF-<k>B p65 levels were higher than in controls, but were not modified in response to paricalcitol (Figures 9A and 9B). However, paricalcitol inhibited TWEAk-induced NF-κB2 activation in the kidney, as evidenced by reduced nuclear p52 and RelB accumulation in tubular epithelial cells. (Figures 9C and 9D). Furthermore, paricalcitol inhibited the DNA binding activities of p52 and RelB induced by TWEAK (Figure 9E). In this sense, TWEAK activation of the NF-<k>B2 pathway in tubular cells leads to the synthesis of specific chemokines, such as CCL21A and CCL19. In mice injected with TWEAK, paricalcitol reduced the expression of the kidney genes CCL21A and CCL19 (Figure 10A) and CCL21 protein levels (Figure 10B). Interestingly, CCL21 localized to the renal tubules in TWEAK-injected mice (Figures 10C and 10D), i.e., in the same renal structures where the active NF-kB2 components RelB and p52 were located (Figure 9C). These data suggest that the anti-inflammatory effects of paricalcitol could be mediated by modulation of NF-<k>B2 target genes.

In TWEAK-injected mice, inhibition of the NF-κB2 pathway with SN52, a synthetic peptide that specifically blocks nuclear translocation of the p52/NF-κB2 subunit (Figures 11A and 11B), significantly decreased the number of monocytes/macrophages. infiltrators (F4/80+ cells) and T lymphocytes (CD3+) (Figures 8A and 8B), thus showing a clear anti-inflammatory effect. Interestingly, in TWEAK-injected mice, SN52 prevented the upregulation of several proinflammatory genes, including the NF-<k>B2-specific target genes CCL21A and CCL19 (Figure 8E), and are known to also be regulated by NF-<k>B2. k>B1 (IL-6, CCL2, and CCL5) (Figure 8C). These data clearly demonstrate that NF-kB2 inhibition could be a potential therapeutic option for renal inflammatory diseases.

• Paricalcitol inhibits the non-canonical NF-kB2 pathway in experimental unilateral ureteral obstruction.

In unilateral ureteral obstruction (UUO model), paricalcitol decreases the infiltration of renal inflammatory cells and the expression of CCL5 and CCL2, without blocking the nuclear translocation of p65/NF-KB1. Therefore, we investigated whether paricalcitol modulated the NF-<k>B2 pathway in the UUO. Paricalcitol prevented the increase in RelB levels and its nuclear localization observed in obstructed kidneys (Figure 12A). Furthermore, it blocked pl0o/p52 processing and p52 nuclear translocation and DNA binding activity (Figures 12A, 12B). In contrast, NF-kB1 activation, assessed by lKB-a phosphorylation and p65/NF-KB DNA binding activity, was not modified in obstructed kidneys treated with paricalcitol (Figure 12C). Importantly, in obstructed kidneys, paricalcitol prevented the infiltration of inflammatory cells and the increased expression of proinflammatory factors, such as CCL2, CCL5, IL6 (Figure 13B) and specific chemokines regulated by NF-kB2, including CCL21A and CCL19 (Figure 13C).

• Paricalcitol inhibits the NF-kB2 pathway, but not the NF-kB1 pathway, in folic acid-induced experimental acute kidney injury.

Next, we investigated the effect of paricalcitol and the relative contribution of the NF-<k>B2 pathway to folic acid (FA)-induced kidney damage in mice. Paricalcitol alone blocked NF-kB2 activation, as determined by RelB and p52 levels, nuclear translocation and DNA binding activity of RelB (Figures 14A, 14B, 14E), and gene and protein expression of the NF-kB2-dependent cytokines, such as CCL21A, as well as other proinflammatory factors (Figures 14F and 14G), while the activation of NF-kB1, assessed by lKB-a phosphorylation, was not modified (Figure 14C). Importantly, paricalcitol also prevented the infiltration of renal inflammatory cells (Figure 15). These data suggest that paricalcitol protects against folic acid-induced kidney injury by inhibiting inflammation and the non-canonical NF-kB2 pathway. Furthermore, paricalcitol also restored changes in kidney function assessed by serum BUN levels (Figure 14H), showing the beneficial effect of this VDRA in the acute phase of kidney damage in this experimental model of kidney injury.

Example 2.10. Paricalcitol inhibits the NF-kB2 pathway independently of MARSS

To test the hypothesis that MARSS could be a receptor for VDRA, MARSS was blocked using a gene silencing approach. HK2 cells were transfected with a MARSS siRNA or a control siRNA and then pretreated or not with paricalcitol before TWEAK stimulation. In MARSS-silenced cells, paricalcitol decreased TWEAK-induced NF-kB2 activation, suggesting a MARSS-independent effect of paricalcitol (Figure 16).

Claims (7)

i.In vitro method to detect kidney disease that includes: to. determine at least the level of TRAF3 protein in peripheral blood mononuclear cells (PBMC) isolated from blood samples obtained from the patient, b. wherein if the level determined in step (a) is statistically lower than the level determined in healthy control subjects, this is an indication that the patient suffers from kidney disease. 2. In vitro method according to claim 1, wherein the kidney disease manifests clinically as acute kidney injury (AKI) or chronic kidney disease (CKD), or the kidney disease has progressed to end-stage renal disease (ESRD). 3. In vitro method to decide or recommend whether patients suffering from kidney disease should be treated with analogues or derivatives of vitamin D that includes: to. determine at least the level of TRAF3 protein in peripheral blood mononuclear cells (PBMC) isolated from blood samples obtained from the patient, b. wherein if the level determined in step (a) is statistically lower than the level determined in healthy control subjects, treatment with vitamin D analogues or derivatives is recommended. 4. In vitro method to predict the response of patients suffering from kidney disease to treatment with analogues or derivatives of vitamin D that includes: to. determine at least the level of TRAF3 protein in peripheral blood mononuclear cells (PBMC) isolated from blood samples obtained from patients before administration of the vitamin D analogue or derivative, b. wherein if the level determined in step (a) is statistically lower than the expression level determined after administration of the vitamin D analogues or derivatives, this is an indication that the patient responds to the treatment. 5. In vitro method according to any of claims 3 or 4, wherein the kidney disease manifests clinically as acute kidney injury (AKI) or chronic kidney disease (CKD), or the kidney disease has progressed to end-stage renal disease (ESRD). 6. In vitro method according to any of claims 3 to 5, wherein the analogue or derivative of vitamin D is selected from the group comprising: paricalcitol, calcitriol, calcidiol, alfacalcidol, tacalcitol, calcifediol, calcipotriol, maxacalcitol, doxercalciferol and/or falecalcitriol. 7. In vitro use of TRAF3 protein level determined in peripheral blood mononuclear cells (PBMC) isolated from blood samples obtained from the patient to detect kidney disease, to predict the response of patients suffering from kidney disease to treatment with analogues or derivatives of vitamin D, or to decide or recommend whether patients suffering from kidney disease should be treated with vitamin D analogues or derivatives.